Nederlandse samenvatting
De behandeling van orgaandonoren met dopamine gaat gepaard met een significante verbetering van de initiële transplantaat functie bij ontvangers van een nier of hart transplantaat. NOD is een synthetische N-acyl dopamine ontwikkeld voor gebruik bij orgaandonoren met als doel een hydrofoob dopamine derivaat te creëren dat geen invloed heeft op de systemische bloeddruk. Door zijn ontstekingsremmende, nier beschermende en cel protectieve eigenschappen is NOD een interessante kandidaat voor klinisch gebruik in settings van ischemie geinduceerde acute nierbeschadiging. Het belangrijkste doel van dit proefschrift was het onderzoeken van relevante chemische en biologische eigenschappen die NOD zijn nier beschermende effecten verlenen. We bestudeerden het vermogen van NOD om TRPV1 te activeren en het prominente redox-actieve karakter, dat het redoxmilieu binnen de intracellulaire compartimenten kan veranderen.
De experimentele studies beschreven in dit proefschrift werden uitgevoerd, ten eerste om aan te tonen dat NOD bescherming biedt in een model van ischemie-geïndu- ceerde acute nier schade, ookwel aute kidney injury (AKI) genoemd, via TRPV1 activering. Ten tweede om de moleculaire entiteiten binnen NOD af te bakenen die nodig zijn voor TRPV1 activering en voor zijn ontstekingsremmende eigenschappen. Ten derde om de invivo weefseldistributie en eliminatiekinetiek van NOD te bestuderen en ten vierde om aan te tonen dat de redoxactiviteit van NOD het celgedrag significant beïnvloedt, d.w.z. dat NOD ER stress en UPR induceert. Het potentiële de potentiële mogelijkheden voor het gebruik van NAD’s in de transplatatiegeneeskunde wordt besproken in hoofdstuk 2.
In hoofdstuk 3 wordt het nier beschermende effect van NOD in een setting van warme ischemie-geinduceerde AKI onderzocht. We hebben aangetoond dat de behande- ling met NOD de nierfunctie bij warme ischemie-geïnduceerde AKI verbetert, zelfs wan- neer de behandeling na het optreden van de nier ischemie wordt gestart. Een verbeterde nierfunctie ging niet gepaard met grote veranderingen in de nierperfusie. Wij hebben aangetoond dat de nier beschermende eigenschappen van NOD via TRPV1 plaatsvindt. NOD behandeling van ontvanger ratten na niertransplantatie leidt niet tot een verbete- ring van de nierfunctie in vergelijking met onbehandelde ontvanger ratten. Het gebrek aan een therapeutisch effect van NOD bij ontvanger ratten kon niet worden verklaard door het verlies van TRPV1 – functionaliteit.
In hoofdstuk 4 hebben we de chemische entiteiten binnen NOD onderzocht die TRPV1 activeren en verantwoordlijk zijn voor de ontstekingsremmende effecten. Door het veranderen van de aromatische-, amide-linker-en vetzuurgroepen van NOD hebben we nieuwe verbindingen gesynthetiseerd. Met behulp van calcium microfluorimetrie hebben we een EC50 waarde van 12,7µM voor TRPV1 activering door NOD gemeten. Acetylering van de orthodihydoxygroepen (geacetyleerd (A-) NOD) of verkorting van de amide-linker (ΔNOD) verminderde de effectiviteit voor TRPV1 activering. Een verdere afname van het effect van ΔNOD werd gevonden de positie van de carbonyl-en amide- groepen te verwisselen. De afwezigheid van één hydroxylgroep aan de aromatische ring (N-octanoyl tyramine (NOT)) of het gebruik van een vertakte vetzuur als esterfunctie
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(N-pivaloyl dopamine (NPiD)) resulteerde in een significante afname van de TRPV1-ac- tivatie. Docking studies en site-directed mutagenese hebben soortgelijke ankerresiduen voor interacties van capsaïcine of NOD met TRPV1 aan het licht gebracht. Echter,een mutatie van arginine naar alanine op positie 491 (R491) had alleen invloed op TRPV1 acti- vering door NOD maar niet door capsaïcine. Met uitzondering van ΔNOD is de aanwezig- heid van een intacte catecholstructuur een voorwaarde voor TNF-α induceerde remming van VCAM-1 en voor de inductie van heem oxygenase-1 (HO-1) expressie. NPiD en A-NOD vertonen beide ontstekingsremmende eigenschappen vergelijkbaar met NOD.
In hoofdstuk 5 hebben we de radiotracer [18F] F-NOD ontwikkeld om de in vivo eliminatiekinetiek van NOD te bestuderen. Eerst hebben we aangetoond dat de biologi- sche eigenschappen van F-NOD en NOD niet van elkaar verschillen, d.w.z. beide remmen de TNF-α-geïnduceerde VCAM-1 expressie, beide induceren de expressie van HO-1, beide beschermen de endotheelcellen tegen koud-geinduceerde schade en beide active- ren TRPV1. [18F]F-NOD kan efficiënt radioactief gelabeld en is stabiel inratten plasma en fosfaatbuffer. In vivo wordt [18F]F-NOD snel via het nier-en hepatobiliaire systeem geelimineerd.
Omdat redoxcondities tussen intracellulaire compartimenten verschillen, en NOD zeer éénvoudig in een greduceerde als ook in een geoxideerde vorm over kan gaan, bekeken we in hoofdstuk 6 of synthetische N-acetyl dopamines (NAD's), ER stress bewerkstelligt met als consequentie dat de zogenaamde unfolded protein response (UPR) geinduceerd wordt. Deze eigenschap van NAD’s lijkt afhankelijk te zijn van de redox-activiteit van deze verbindingen. In niet toxische concentratries remt NOD de celproliferatie van HUVEC's sterk, zeer waarschijnlijk door een vertraging in cell cyclus progressie zodat de cellen in de S-G2/M-fase va de cell cyclus blijven. In overeenstemming hiermee werd gevonden dat de expressie van verschillende genen, betrokken bij de S-G2/M-progres- sie, door NOD aanzienlijk verlaagd worden. Interessant is dat behandeling met NOD op lange termijn resulteerde in hypo-metabolisme en thermotolerantie, zoals gesuggereerd wordt door een verminderde intracellulaire ATP-concentratie, activering van AMPK en een verhoogde weerstand tegen de kou toegebrachte celbeschadiging.
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Acknowlegements
At times our own light goes out and is rekindled by a spark from another person. Each of us has cause to think with deep gratitude of those who have lighted the flame within us—Albert Schweitzer.
I take this moment to thank everyone who has helped me endure this journey and help me realise this dream.
First and foremost a special thanks and appreciation goes to my enthusiastic super- visor, Prof. Dr. Benito Yard. My PhD has been an amazing experience and I thank Benito wholeheartedly, not only for his tremendous academic support, but also for giving me so many wonderful opportunities like interaction with university of Groningen and par- ticipation in transplantation Summer School Groningen. I am thankful to him for his patience, dedication and ingenious teaching techniques.
I am thankful to Prof. Dr. Mathias Hafner, for his valuable advices during my doctoral thesis endeavour for past four and half years. His observation and comments helped me moved ahead in investigational path in depth. I am thankful to him for intro- ducing me to many new and productive collaboration opportunities. I deeply appreciate your help with travel and living cost for attending San Francisco WTC: I am thankful to Albert und Anneliese Konanz-Stiftung, from Hochschule Mannheim for providing me scholarship.
I humbly thank Marco. C. Harmsen for accepting me as his PhD student and for providing me this opportunity. For kindess, patience ,constant encouragement and sup- port that I have received from him. I am very thankful to his constant input with writing of the thesis and optimism with which he always bolstered my confidence. I also highly thankful for several inspirational discussions.
I am grateful to Prof. Dr. med. Hans-Peter Hammes for providing me opportunity to be part of the GRK880/3–4, Vascular Medicine Graduate School and for organizing various interesting scientific lectures and the financial support during initial phase of my PhD studies.
I thank Prof. Dr. med. Bernhard Krämer, Director of Nephrology, for providing me a chance to be part of Nephrology Department.
Thanks to Prof. Dr. med. Rolf-Detlef Treede for collaboration and for providing access to the calcium imaging facility. I am thankful to Dr. Wolfgang Greffrath and Dr. Uta Binzen for support and trust they have showed in me and for wonderful learning experience. I am thankful to Mrs. Handan Morez and Mrs. Ulrike Horst for introducing me to calcium imaging technique which proved to be a great research tool in the study.
I am grateful to Dr. Marc Pretze and Dr. Ralf Losel, who have helped me transform basic ideas and mere chemical structures from Chemdraw screens into reality by syn- thesis. I also thank Dr. Marc Pretze for numerous useful discussions which provided breakthrough in understanding of NADs behaviour. I am highly grateful to Marc for helping in the tumultuous times and standing by me not only as a supporting collab-
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orator but also providing insight in chemical experimental work. I am thankful to Dr. Bastian Theisinger and Novaliq GmbH for support with production of NOD and other
N-Acyl dopamine derivatives.
I thank Dr. Sarah Klotz for helping with the animal experiments, for collaboration and profound scientific discussions.
I would like to express my sincere thanks to Dr. Julio Caballero for generously shar- ing his time and expert knowledge on molecular docking. With his help TRPV1-NADs molecular model was established and which proved to be great leap in helping me under- stand the interaction of TRPV1 and NADs and wet lab data.
I am grateful to Dr. Christin Saur from Department of Pathology, University Hospi- tal Mannheim Medical faculty Mannheim, University of Heidelberg for his help with the sequencing of the mutants, which saved not only saved funds but also time.
I would like to thank present and past members of the Prof. Yard’s working group, Mrs. Annette Breedijk, Mrs. Katharina Prem, Mrs. Renate Stein, Mrs. Silke Deiters, and Dr. Sarah Klotz for technical support, friendly conversation and for proving a very friendly and independent working environment. I thank Dr. Shiqi Zhang, Dr. Eleni Sta- mellou and Dr. Johannes Wedel for useful discussion and collaborations. I thank Danfeng Zhang, Yinchung Li, Angelica Rodrigues, Björn Hofmann, Diego Pastene, Sara Medina Balbuena, Lovis Kling, Jana Braun, and Thomas Albrecht for the friendly and organised working environment.
My special thanks goes to Hilda van den Bos, Kathrin Hammon, Pietro Marcato, Igor Dolgowjasow, Jonas Heckeli and Isabel Ann Wedenig. Teaching is a part of learning and the privilege to supervise them during their interships, Bachelor or Master thesis in the lab has been an enriching learning experience.
My heartily thanks go to Annette Breedijk, Sarah Klotz and Marianne Heitzmann for the delightful and amicable conversation in the office. I found many insights and new ways of thinking. In lows, I found my high, and in high I still could survive, for you were there and I am thankful to thy.
My sincere thanks go to the neighbouring lab and their members who have rendered their help several occasions. I am thankful to Ms. Jutta Schulte, Dr. Sonia Velasquez, Ms. Miriam Bierbaum, and Ms. Nadine Dietrich for lending a helping hand.
I am also grateful to Ms. Ariane Tomsche from HS Mannheim for her support every now and then with techniques, troubleshooting and help with bureaucracy.
I also want to thank my fellow scientist friends from India Dr. Nagesh Shanbhag, Dr. Siladitta Biswas, Dr. Anupriya Chatterjee for their company and constructive discussions which have brought me forward many times and help me clear views on many topics.
My heartfelt thanks go to Dr. Linda Hartmann, Dr. Paulina Nunez, Dr. Kristina Mauer, Dr. Daniela Rosenberg, Dr. Juan Juan Zhang, Dr. Sabrina Voss, Dr. Jagriti Srivas- tava, and Ms. Handan Morez for their camaraderie and company, making the non-sci- entific part of my thesis colourful. I am also thankful to Mrs. Jenni Maier and Mr. Oliver Kresten for the friendship and affection they have provided. My sincere thanks go to
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Dr. Martin Dittmer, who has been one of my best friends and helped me out with many things. I thank you for the care and positive mind-set that you have lent in several of occasions.
A note of thanks also goes to Prof. Dr. Kunal Mukhopadhyay from the Department of Bioengineering Birla institute of Technology, Mesra Ranchi and Prof. Dr. H. Devaraj from the Department of Zoology, University of Madras who have helped me with my first steps in research.
Finally, I thank my parents who have always been inspiration and great support throughout my life. I thank you for your patience and optimism in all of my adventures. Your words always made it bright in darkest of the night and I could keep my equipoise in many tempestuous situations. I am also grateful to my two lovely siblings; Chetna and Utkarsh, who have been my best friends and are sunshine in my life, supporting me through ups and downs.
I am very thankful to Ram, who I met towards the completion of my work, who cheered me up and supported me in all possible ways to make me reach the end line. He anchored me to hope when I saw none.
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Abbreviations
Abbreviation Full Form
([Ca + + ]i) Cytoplasmic calcium
[18F] Fluorine-18 radioisotope ° C Degree centigrade
µ Micro
3-MT 3-methoxytyramine
A Alanine
ADP Adenosine diphosphate AKI Acute kidney injury Allo Allogeneic
AMP Adenosine monophosphate AMPK 5' AMP-activated protein kinase A-NOD Acteylated NOD
AP-1 Activator protein 1
ARE Antioxidant response element ASL Arterial spin labelling
ATF6 Activating transcription factor 6 ATP Adenosine triphosphate BBr3 Boron tribromide
BD Braindead
Bq Becquerel, unit of radioactivity BSA Bovine serum albumin C/EBP C/EBP homologous protein Ca + 2 Calcium ion
CaCl2 Calcium chloride cAMP Cyclic AMP
CAP Capsaicin
CAP-OH (E)-N-(3,4-dihydroxybenzyl)-8-methylnon-6-enamide CB1 Cannabinoid receptor type 1
CB2 Cannabinoid receptor type 2 cDNA Complementary DNA CGRP Calcitonin gene-related peptide CH2Cl2 Dichloromethane
Ci Curie, non-SI unit of radioactivity CMV Cytomegalovirus
Appendix 175 Abbreviation Full Form COX-2 Cyclooxygenase-2 COX-2 Cyclooxygenase-2 CPZ Capsazepine d.c. Decay counted DA Dopamine
DGF Delayed graft function DHA Docosahexaenoic acid DMF Dimethylformamide DMSO Dimethyl sulfoxide DRG Dorsal root ganglion DTT Dithiothreitol
e- Eletron
EC50 Effective concentration 50
ED1 Ectodysplasin A
EDTA Ethylenediaminetetraacetic acid EPA Eicosapentaenoic acid ER Endoplasmic reticulum
ERSE Acting ER-stress response element Et2O Petroleum ether
F Phenylalanine
FAIR Flow-sensitive alternating inversion recovery FBS Fetal bovin Serum
FCS Fetal calf Serum
FDA Food and Drug Administration F-NOD 2-fluoro-N-octanoyl dopamine
g Gram
GAPDH Glyceraldehyde 3-phosphate dehydrogenase H + Proton
H2O Water
H2O2 Hydrogen peroxide H2S Hydrogen sulphide
HEK293 Human Embroynic Kidney cells 293
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HO-1 Heme oxygenase-1
HPLC High performance liquid chromatography
hr Hour
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176
Abbreviation Full Form
HUVECs Human umbilical vein endothelial cells
Hz Hertz
I Isoleucine
I/R-injury Ischemia reperfusion injury ICAM1 Intercellular adhesion molecule 1) IL-6 Interleukin 6
IPC Ischemic preconditioning IRE1 Inositol requiring kinase
JNK1 C-jun N-terminal protein kinase 1 (JNK1)
k Kilo K + Potassium ion K2CO3 Potassium carbonate KCl Potassium chloride l Liter L Leucine LDH Lactate dehydrogenase LiAlH4 Lithium aluminium hydride
LOX Lysyl oxidase) LT Leukotrienes
M Molar
M Methionine
m Mili
MALDI-TOF Matrix-assisted laser desorption/ionization-time of flight MAPK Mitogen-activated protein kinase
MeCN Acetonitrile MgCl2 Magnesium chloride MgSO4 Magnesium sulfate
min Minute
mm Milimeter
MOI Multiplicity of infection MRI Magnetic resonance imaging mRNA Messenger RNA
n Nano
N Asparagine
Na + Sodium ion
NAANs N-acyl conjugates of amino acids and neurotransmitters NaCl Sodium chloride
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Abbreviation Full Form
NADA N-arachidonoyl dopamine NADH Nicotinamide adenine dinucleotide NADPH Reduced form of NADP
NADs N-acyl dopamines NaHCO3 Sodium bicarbonate
NFAT Nuclear Factor of Activated T-cells NF-κB Nuclear factor κb
nm Nanometer
NMR Nuclear magnetic resonance NO Nitrogen oxide
NOD N-octanoyl dopamine NOT N-octanoyl tyramine NPiD N-pivaloyl dopamine
Nrf-2 Nuclear factor erythroid 2-related factor 2 OLDA N-oleoyl dopamine
PBS Phosphate-buffered saline PDI Protein disulfide isomerase PERK (PKR)-like ER kinase
PET Positron-emission tomography PG Prostaglandins
PMNs Polymorphonuclear leukocyte
PPAR Peroxisome proliferator-activated receptors ppm Parts per million
PVDF Polyvinylidene fluoride
QPCR Quantitative polymerase chain reaction
R Arginine
RCY (Radiochemical yield RNA Ribonucleic acid ROI Regions of interest
RT PCR Reverse transcription polymerase chain reaction rTRPV1 Rat TRPV1
S Serine
s Seconds
SAM S-adenosyl-l-methionine SD Standard deviation sd Sprague Dawley rat SEM Standard error of mean
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Abbreviation Full Form
SP Substance P
SUVbw Standardized uptake value based on body weight
Syn Syngeneic
T Threonine
TAK1 Transforming growth factor β-activated kinase 1 TBS Tris-buffered saline
TFA Trifluoroacetic acid THF Tetrahydrofuran TI Inversion time
TLC Thin-layer chromatography TNF Tumor necrosis factor
TRPV1 Transient receptor potential channel of the vanilloid receptor subfamily, subtype 1 Tx Transplantation
UPR Unfolded protein response
UV Ultraviolet
VCAM1 Vascular cell adhesion protein 1 VDW Van der Waals force
WT Wild type
Xbp1 X-box-binding protein 1
Y Tyrosine
ΔNOD Delta NOD
ΔNODR Delta NOD reverse DNA Deoxyribonucleic acid
Appendix 179
Author affiliations
Anna-Isabelle Kälsch1 Benito. A. Yard1 Bernhard K. Krämer 1 Björn B. Hofmann 1 Björn Wängler2 Carmen Wängler3 Charalambos Tsagogiorgas4 Eleni Stamellou1 Fabian Zimmer 6 Frank G. Zöllner 6 Handan Morez7 Jakob Walter8 Johannes Wedel1 Julio Caballero9 Marc Pretze2 Mareike Roscher2 Martin.C. Harmsen10 Mathias Hafner11 Rolf-Detlef Treede7 Sarah Klotz 1 Simone Hoeger1 Uta Binzen7 Wolfgang Greffrath7 Yingchun Li 11: V Department of Medicine, University Hospital Mannheim, Medical Faculty Mannheim, Heidelberg University
2: Molecular Imaging and Radiochemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim,
Heidelberg University, Mannheim, Germany
3: Biomedical Chemistry, Department of Clinical Radiology
and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University 4: Department of Anaesthesiology and Intensive Care Medicine,
University Hospital Mannheim, Mannheim, Germany 5: Experimental Dermatology, Medical Faculty Mannheim,
Heidelberg University, Mannheim, Germany.
6: Computer Assisted Clinical Medicine, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
7: Division of Neurophysiology, Centre of Biomedicine and Medical Technology Mannheim (CBTM), Medical Faculty Mannheim, Heidelberg university, Mannheim, Germany
8: Bayer Healthcare Pharma AG, Berlin, Germany 9: Center for Bioinformatics and Molecular Simulations,
Faculty of Engineering in Bioinformatics, Universidad de Talca, Talca, Chile 10: University of Groningen, University Medical Centre Groningen,
Department of Pathology and Medical Biology, Groningen, The Netherlands 11: Institute for Molecular and Cellular Biology,